Caracterização molecular de estirpes de Aeromonas spp. resistentes a antibióticos beta-lactâmicos

Mestrado em Microbiologia MolecularEstudos feitos em todo o mundo têm demonstrado que Aeromonas spp. estão distribuídas universalmente e podem ser isoladas de amostras clínicas, alimentares e ambientais. São exemplo de microrganismos patogénicos emergentes. Em sistemas de aquacultura, as infecções bacterianas são a causa principal das quebras de produtividade. O uso intensivo de antibióticos beta-lactâmicos como agentes terapêuticos levou ao aparecimento de estirpes multiresistentes. Neste trabalho, foram analisadas estirpes de Aeromonas isoladas de amostras de tecido da pele e rins de trutas arco-íris (Oncorhynchus mykiss) de uma aquacultura da Universidade de Trás-os-Montes e Alto Douro. Foram incluídas também estirpes de referência. Os testes de susceptibilidade a antibióticos beta-lactâmicos revelaram que a maior parte dos isolados é resistente a penicilinas e cefalosporinas de 1ª geração. Em 7 isolados foi detectada resistência a carbapenemos (imipenemo). Por forma a caracterizar o mecanismo de resistência dos isolados resistentes ao imipenemo, foram desenhados "primers" para amplificar por PCR genes de resistência homólogos a cphA e/ou imiS, que codificam as metalo-beta-lactamases CphA e ImiS, já descritas em Aeromonas spp.. Em 5 dos isolados e em 2 estirpes referência, foi possível amplificar por PCR, com os "primers" desenhados, Aer- R e Aer-F, um fragmento com o tamanho esperado, cerca de 670 pb, a partir do DNA total das estirpes de Aeromonas spp.. A análise das sequências nucleotídicas e das sequências de aminoácidos deduzidas revelou tratarem-se de fragmentos de DNA de genes homólogos a cphA/imiS, que codificam para metalo-beta-lactamases de classe B, subclasse B2. Procedeu-se também à detecção de sequências homólogas por hibridação DNA-DNA, usando uma sonda marcada por PCR com os "primers" Aer-R e Aer-F. Ocorreu hibridação entre o fragmento de DNA usado como sonda e o DNA de todos os isolados que apresentavam actividade de carbapenemase. Pretendeu-se, também, caracterizar os isolados ao nível molecular, através de técnicas de tipagem genética como ARDRA e rep-PCR. Os perfis ARDRA obtidos por digestão com MboI e AluI permitiram identificar os isolados como Aeromonas bestiarum Aeromonas hydrophila e Aeromonas media. Os perfis REP- e BOX-PCR resultantes mostraram-se mais complexos que os perfis ARDRA e permitiram avaliar o grau de diversidade genómica dos isolados, mostrando-se úteis na tipagem genética de isolados deste grupo de procariotas.Worldwide studies have demonstrated that Aeromonas spp. are universally distributed and widely isolated from clinical, environmental and food samples. They are an example of emerging bacterial pathogens. In aquaculture explorations, bacterial infectious diseases are the major cause of decreases in productivity. The extensive use of beta-lactam antibiotics as therapeutic agents has generated multiresistant strains. In this work, tissue samples from the skin and kidney of rainbow trouts (Oncorhynchus mykiss) from a fish farm at the Universidade de Trás-os-Montes e Alto Douro were examined for the presence of Aeromonas spp. Reference strains were also included in this study. The susceptibility to antibiotics tests revealed that most of these strains are resistant to penicillins and 1st generation cephalosporins. It was detected resistance to carbapenems (imipenem) in 7 aeromonads. In order to understand the resistance mechanism of the strains that showed resistance to carbapenems, primers Aer-R and Aer-F were designed to detect by PCR, homologous genes to cphA/imiS, which code for the metallo-beta-lactamases CphA and ImiS, already described in Aeromonas spp. In 5 isolates and in 2 reference strains, it was possible to amplify by PCR a DNA fragment with 670 bp as expected, whose sequence was determined. The nucleotide and deduced amino acid sequence analysis revealed that these are DNA fragments homologous to cphA/imiS, that code for class B, subclass B2 metallo-betalactamases. DNA-DNA hybridization was also applied using a DNA fragment, amplified by PCR with primers Aer-R and Aer-F, as probe. There was hybridization between the probe and the DNA from all the strains that showed resistance to carbapenems. Aeromonads isolates were also compared using different molecular typing techniques like ARDRA and rep-PCR. ARDRA profiles obtained from enzymatic digestion of rDNA 16S wilth MboI and AluI, allowed the identification of strains as Aeromonas bestiarum, Aeromonas hydrophila and Aeromonas media. Fingerprinting by REP- and BOX- PCR provided complex genomic profiles, proving that these are good methods for typing aeromonads

Resistência a antibióticos de último recurso em ambientes naturais

Doutoramento em BiologiaLast-resort antibiotics are the final line of action for treating serious infections caused by multiresistant strains. Over the years the prevalence of resistant bacteria has been increasing. Natural environments are reservoirs of antibiotic resistance, highly influenced by human-driven activities. The importance of aquatic systems on the evolution of antibiotic resistance is highlighted from the assumption that clinically-relevant resistance genes have originated in strains ubiquitous in these environments. We hypothesize that: a) rivers are reservoirs and disseminators of antibiotic resistance; b) anthropogenic activities potentiate dissemination of resistance to last-resort antibiotics. Hence, the main goal of the work is to compare the last-resort antibiotics resistome, in polluted and unpolluted water. Rivers from the Vouga basin, exposed to different anthropogenic impacts, were sampled. Water quality parameters were determined to classify rivers as unpolluted or polluted. Two bacterial collections were established enclosing bacteria resistant to cefotaxime (3rd generation cephalosporin) and to imipenem (carbapenem). Each collection was characterized regarding: phylogenetic diversity, antibiotic susceptibility, resistance mechanisms and mobile genetic elements. The prevalence of cefotaxime- and imipenem-resistant bacteria was higher in polluted water. Results suggested an important role in the dissemination of antibiotic resistance for Enterobacteriaceae, Pseudomonas and Aeromonas. The occurrence of bacteria resistant to non-beta-lactams was higher among isolates from polluted water as also the number of multiresistant strains. Among strains resistant to cefotaxime, extended-spectrum beta-lactamase (ESBL) genes were detected (predominantly blaCTX-M-like) associated to mobile genetic elements previously described in clinical strains. ESBL-producers were often multiresistant as a result of co-selection mechanisms. Culture-independent methods showed clear differences between blaCTX-M-like sequences found in unpolluted water (similar to ancestral genes) and polluted water (sequences identical to those reported in clinical settings). Carbapenem resistance was mostly related to the presence of intrinsically resistant bacteria. Yet, relevant carbapenemase genes were detected as blaOXA-48-like in Shewanella spp. (the putative origin of these genes), and blaVIM-2 in Pseudomonas spp. isolated from polluted rivers. Culture-independent methods showed an higher than the previously reported diversity of blaOXA-48-like genes in rivers. Overall, clear differences between polluted and unpolluted systems were observed, regarding prevalence, phylogenetic diversity and susceptibility profiles of resistant bacteria and occurrence of clinically relevant antibiotic resistance genes, thus validating our hypotheses. In this way, rivers act as disseminators of resistance genes, and anthropogenic activities potentiate horizontal gene transfer and promote the constitution of genetic platforms that combine several resistance determinants, leading to multiresistance phenotypes that may persist even in the absence of antibiotics.Antibióticos de último recurso são usados no tratamento de infecções graves causadas por estirpes multiresistentes. A prevalência de bactérias resistentes a estes antibióticos tem aumentado. Os ambientes naturais, influenciados pela actividade humana, são reservatórios de bactérias resistentes e de genes de resistência. Vários genes de resistência com grande impacto na clínica têm presumivelmente origem em estirpes ubíquas em sistemas aquáticos, o que realça a importância destes ambientes na evolução de resistência. Este estudo assenta nas seguintes hipóteses: a) os rios são reservatórios e disseminadores de resistência a antibióticos; b) as atividades antropogénicas potenciam a disseminação de resistência a antibióticos de último recurso nestes ambientes. Assim, foi estabelecido como objectivo comparar o resistoma ambiental referente a antibióticos de último recurso, em rios poluídos e não poluídos. Foram amostrados rios na Bacia Hidrográfica do Vouga, expostos a diferentes impactos antropogénicos. Os rios foram classificados como poluídos e não poluídos de acordo com parâmetros de qualidade da água. Duas colecções foram estabelecidas: bactérias resistentes a cefotaxima (cefalosporina de 3ª geração) e a imipenemo (carbapenemo). Cada colecção foi caracterizada em termos de diversidade filogenética, susceptibilidade a antibióticos, mecanismos de resistência e elementos genéticos móveis. A prevalência de bactérias resistentes foi superior em águas poluídas. Os resultados sugerem que nestes ambientes Enterobacteriaceae, Pseudomonas e Aeromonas têm um papel importante na disseminação de resistência. Os níveis de resistência a não beta-lactâmicos foram superiores em águas poluídas, assim como o número de estirpes multiresistentes. Detectaram-se genes de beta-lactamases de espectro alargado, associados a elementos genéticos móveis previamente descritos em isolados clínicos. Métodos independentes do cultivo revelaram diferenças claras entre a diversidade de sequências do tipo blaCTX-M em rios poluídos (idênticas às encontradas em isolados clínicos) e não poluídos (similares a genes ancestrais). A resistência a carbapenemos foi maioritariamente relacionada com a presença de bactérias intrinsecamente resistentes. No entanto, foram identificados genes de carbapenemases relevantes tais como blaOXA-48 em Shewanella spp. (origem putativa destes genes) e blaVIM-2 em Pseudomonas spp. de rios poluídos. Métodos independentes do cultivo mostraram que, nestes rios, a diversidade de genes similares a blaOXA-48 é superior ao que tem sido relatado. Detectaramse diferenças evidentes entre rios poluídos e não poluídos, em termos de prevalência, diversidade filogenética e susceptibilidade a antibióticos em bactérias resistentes e ocorrência de genes de resistência clinicamente relevantes. Estes dados validam as hipóteses colocadas. Assim, estes sistemas aquáticos actuam como reservatórios de genes de resistência. As actividades antropogénicas potenciam a disseminação destes genes e a constituição de plataformas genéticas complexas, originando fenótipos de multiresistência que poderão persistir mesmo na ausência de antibióticos

Recycling of marine aquaculture wastewater using a microalgae-bacterial granular sludge system

Aquaculture has become the fastest growing animal food-producing sector. In a near future, an intensification of the aquaculture practices is expected to cope with the ever-increasing fish demand. However, for land-based aquaculture farms, this growth implies the capture of higher water volumes from nearby water bodies and, consequently, the discharge of higher volumes of wastewater, containing organic carbon, nutrients, and often recalcitrant pollutants (e.g. pharmaceuticals). The expansion of the land-based aquaculture sector is currently offset due to the lack of space and water supplies, but also due to environmental concerns. Therefore, there is a need for innovative wastewater treatment systems able to reduce energy input, to improve resource use and to reduce the environmental impact. In the present study, microalgae-bacterial granules were developed from a phototrophic microbial consortium autochthonous to the water streams of a marine aquaculture facility. The granular biomass was able to efficiently treat marine aquaculture streams, even when sporadically the antibiotic florfenicol was present, with pollutant reaching levels that allowed water recirculation in fish farms. The ammonium, nitrite, and nitrate concentrations in the treated effluents were below the toxicity limits for marine fish and, the dissolved oxygen levels were within the ideal range for water recirculation. The granules microbial community was dynamic and, its structure was susceptible and adaptable to the changing operational reactor conditions such as the presence of the antibiotic florfenicol. The microbial diversity and functional redundancy within the microbial community seemed to be crucial for the adaptability of the system to the stressors presence. Th symbiosis established between microalgae and bacteria within granules allowed for the effective and environmentally sustainable treatment of marine aquaculture wastewater.info:eu-repo/semantics/publishedVersio

Antibiotic-resistant E.Coli in an UV-treated effluent: Troubled waters ahead?

info:eu-repo/semantics/publishedVersio

Environmental Shewanella xiamenensis strains that carry bla OXA-48 or blaOXA-204 genes: additional proof for bla OXA-48-Like gene origin

This work was supported by Fundação para a Ciência e a Tecnologia (FCT) through grants SFRH/BD/43468/2008 (M.T.) and SFRH/BPD/63487/2009 (I.H.) and by project Phytomarsh (PTDC/AAC-AMB/118873/2010).publishe

Bioremediation of coastal aquaculture effluents spiked with florfenicol using microalgae-based granular sludge – a promising solution for recirculating aquaculture systems

Aquaculture is a crucial industry in the agri-food sector, but it is linked to serious environmental problems. There is a need for efficient treatment systems that allow water recirculation to mitigate pollution and water scarcity. This work aimed to evaluate the self-granulation process of a microalgae-based consortium and its capacity to bioremediate coastal aquaculture streams that sporadically contain the antibiotic florfenicol (FF). A photo-sequencing batch reactor was inoculated with an autochthonous phototrophic microbial consortium and was fed with wastewater mimicking coastal aquaculture streams. A rapid granulation process occurred within ca. 21 days, accompanied by a substantially increase of extracellular polymeric substances in the biomass. The developed microalgae-based granules exhibited high and stable organic carbon removal (83-100%). Sporadically wastewater contained FF which was partially removed (ca. 5.5-11.4%) from the effluent. In periods of FF load, the ammonium removal slightly decreased (from 100 to ca. 70%), recovering 2 days after FF feeding ceased. A high-chemical quality effluent was obtained, complying with ammonium, nitrite, and nitrate concentrations for water recirculation within a coastal aquaculture farm, even during FF feeding periods. Members belonging to the Chloroidium genus were predominant in the reactor inoculum (ca. 99%) but were replaced from day-22 onwards by an unidentified microalga from the phylum Chlorophyta (>61%). A bacterial community proliferated in the granules after reactor inoculation, whose composition varied in response to feeding conditions. Bacteria from the Muricauda and Filomicrobium genera, Rhizobiaceae, Balneolaceae, and Parvularculaceae families, thrived upon FF feeding. This study demonstrates the robustness of microalgae-based granular systems for aquaculture effluent bioremediation, even during periods of FF loading, highlighting their potential as a feasible and compact solution in recirculation aquaculture systems.info:eu-repo/semantics/publishedVersio

Characterization of multi-drug resistant Escherichia coli in the UV-treated outflow of an urban wastewater treatment plant

info:eu-repo/semantics/publishedVersio

Occurrence of carbapenemase-producing Enterobacteriaceae in a Portuguese river: blaNDM, blaKPC and blaGES among the detected genes

Carbapenems are used as last-resort drugs to treat infections caused by multidrug-resistant bacteria. Despite the increasing number of reports of carbapenem-resistant Enterobacteriaceae (CRE), there is still limited information on their distribution or prevalence in the environment. Our aim was to assess the occurrence of CRE in the Lis river (Portugal) and to characterize the genetic platforms linked to carbapenemase genes. We collected six water samples from sites near a wastewater treatment plant (n = 4 samples) and livestock farms (n = 2). Twenty-four CRE were characterized by BOX element-polymerase chain reaction (BOX-PCR), and thirteen representative isolates were analysed by Pulsed-Field Gel Electrophoresis (PFGE) and by sequencing the 16S rRNA gene. Antimicrobial susceptibility testing, PCR screening for carbapenemase-encoding genes, conjugation experiments and plasmid analysis were performed. Four isolates were chosen for whole-genome sequencing. All water samples contained CRE (4.0 CFU/mL on average). Representative isolates were multidrug-resistant (resistant to ciprofloxacin, trimethoprim-sulfamethoxazole and to all β-lactams tested) and were identified as K. pneumoniae, Enterobacter and Citrobacter. Isolates carried plasmids and harboured carbapenemase-encoding genes: blaKPC-3 in K. pneumoniae (n = 9), blaNDM-1 in Enterobacter (n = 3) and blaGES-5 in Citrobacter (n = 1). Conjugation experiments were successful in two Klebsiella isolates. Enterobacter PFGE profiles grouped in one cluster while Klebsiella were divided in three clusters and a singleton. Whole-genome sequencing analysis revealed blaGES-5 within a novel class 3 integron (In3-16) located on an IncQ/pQ7-like plasmid in Citrobacter freundii CR16. blaKPC-3 was present on IncFIA-FII pBK30683-like plasmids, which were subsequently confirmed in all K. pneumoniae (n = 9). Furthermore, blaKPC-3 was part of a genomic island in K. pneumoniae CR12. In E. roggenkampii CR11, blaNDM-1 was on an IncA/C2 plasmid. The carbapenemase-encoding plasmids harboured other resistance determinants and mobile genetic elements. Our results demonstrate that Lis river is contaminated with CRE, highlighting the need for monitoring antibiotic resistance in aquatic environments, especially to last-resort drugs.publishe

Extended Spectrum Beta-Lactamase-Producing Gram-Negative Bacteria Recovered From an Amazonian Lake Near the City of Belém, Brazil

Aquatic systems have been described as antibiotic resistance reservoirs, where water may act as a vehicle for the spread of resistant bacteria and resistance genes. We evaluated the occurrence and diversity of third generation cephalosporin-resistant gram-negative bacteria in a lake in the Amazonia region. This water is used for human activities, including consumption after appropriate treatment. Eighteen samples were obtained from six sites in October 2014. Water quality parameters were generally within the legislation limits. Thirty-three bacterial isolates were identified as Escherichia (n = 7 isolates), Acinetobacter, Enterobacter, and Klebsiella (n = 5 each), Pseudomonas (n = 4), Shigella (n = 3), and Chromobacterium, Citrobacter, Leclercia, Phytobacter (1 isolate each). Twenty nine out of 33 isolates (88%) were resistant to most beta-lactams, except carbapenems, and 88% (n = 29) were resistant to antibiotics included in at least three different classes. Among the beta-lactamase genes inspected, the blaCTX–M was the most prevalent (n = 12 positive isolates), followed by blaTEM (n = 5) and blaSHV (n = 4). blaCTX–M–15 (n = 5), blaCTX–M–14 (n = 1) and blaCTX–M–2 (n = 1) variants were detected in conserved genomic contexts: blaCTX–M–15 flanked by ISEcp1 and Orf477; blaCTX–M–14 flanked by ISEcp1 and IS903; and blaCTX–M–2 associated to an ISCR element. For 4 strains the transfer of blaCTX–M was confirmed by conjugation assays. Compared with the recipient, the transconjugants showed more than 500-fold increases in the MICs of cefotaxime and 16 to 32-fold increases in the MICs of ceftazidime. Two isolates (Escherichia coli APC43A and Acinetobacter baumannii APC25) were selected for whole genome analysis. APC43A was predicted as a E. coli pathogen of the high-risk clone ST471 and serotype O154:H18. blaCTX–M–15 as well as determinants related to efflux of antibiotics, were noted in APC43A genome. A. baumannii APC25 was susceptible to carbapenems and antibiotic resistance genes detected in its genome were intrinsic determinants (e.g., blaOXA–208 and blaADC–like). The strain was not predicted as a human pathogen and belongs to a new sequence type. Operons related to metal resistance were predicted in both genomes as well as pathogenicity and resistance islands. Results suggest a high dissemination of ESBL-producing bacteria in Lake Água Preta which, although not presenting characteristics of a strongly impacted environment, contains multi-drug resistant pathogenic strains

Resistance to Broad-Spectrum Antibiotics in Aquatic Systems: Anthropogenic Activities Modulate the Dissemination of blaCTX-M-Like Genes

We compared the resistomes within polluted and unpolluted rivers, focusing on extended-spectrum beta-lactamase (ESBL) genes, in particular blaCTX-M. Twelve rivers from a Portuguese hydrographic basin were sampled. Physicochemical and microbiological parameters of water quality were determined, and the results showed that 9 rivers were classified as unpolluted (UP) and that 3 were classified as polluted (P). Of the 225 cefotaxime-resistant strains isolated, 39 were identified as ESBL-producing strains, with 18 carrying a blaCTX-M gene (15 from P and 3 from UP rivers). Analysis of CTX-M nucleotide sequences showed that 17 isolates produced CTX-M from group 1 (CTX-M-1, -3, -15, and -32) and 1 CTX-M that belonged to group 9 (CTX-M-14). A genetic environment study revealed the presence of different genetic elements previously described for clinical strains. ISEcp1 was found in the upstream regions of all isolates examined. Culture-independent blaCTX-M-like libraries were comprised of 16 CTX-M gene variants, with 14 types in the P library and 4 types in UP library, varying from 68% to 99% similarity between them. Besides the much lower level of diversity among CTX-M-like genes from UP sites, the majority were similar to chromosomal ESBLs such as blaRAHN-1. The results demonstrate that the occurrence and diversity of blaCTX-M genes are clearly different between polluted and unpolluted lotic ecosystems; these findings favor the hypothesis that natural environments are reservoirs of resistant bacteria and resistance genes, where anthropogenic-driven selective pressures may be contributing to the persistence and dissemination of genes usually relevant in clinical environments